Truncated Cry35 proteins

ABSTRACT

This invention provides truncated Cry35 proteins that surprisingly and unexpectedly have increased pesticidal activity as compared to full-length Cry35 proteins. The subject invention also includes polynucleotides that encode these truncated proteins, transgenic plants comprising a truncated gene of the subject invention, and transgenic plants that produce these truncated proteins. This invention further provides methods of controlling plant pests, including rootworms, with these truncated proteins. The truncated Cry35 proteins of the subject invention are preferably used in combination with Cry34 proteins, which are known in the art. Various surprising advantages of the subject invention will be apparent in light of this disclosure.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to provisional application Ser. No.60/584,324, filed Jun. 29, 2004, the disclosure of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Coleopterans are a significant group of agricultural pests that causeextensive damage to crops each year. Examples of coleopteran pestsinclude corn rootworm and alfalfa weevils. Additional notable examplesinclude Colorado potato beetle, boll weevil, and Japanese beetle.

Insecticidal crystal proteins from some strains of Bacillusthuringiensis (B.t.) are well-known in the art. See, e.g., Höfte et al.,Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteinsare typically produced by the bacteria as approximately 130 kDaprotoxins that are then cleaved by proteases in the insect midgut, afteringestion by the insect, to yield a roughly 60 kDa core toxin. Theseproteins are known as crystal proteins because distinct crystallineinclusions can be observed with spores in some strains of B.t. Thesecrystalline inclusions often comprise a mixture of distinct proteins.

An entirely new insecticidal protein system was discovered in Bacillusthuringiensis as disclosed in WO 97/40162. Unlike the 130 kDa-typeprotoxins, this new system comprises two proteins—one of approximately15 kDa and the other of about 45 kDa. See also U.S. Pat. No. 6,083,499;U.S. Pat. No. 6,127,180; Moellenbeck et al., Nature Biotechnology19:668-672 (2001); and Ellis et al., Applied and EnvironmentalMicrobiology 68:1137-1145 (2002). These proteins have now been assignedto their own classes, and accordingly received the Cry designations ofCry34 and Cry35, respectively. See Crickmore et al. website(biols.susx.ac.uk/home/Neil_Crickmore/Bt/). Many other proteins of thistype of system have now been disclosed. See e.g. U.S. Pat. No.6,372,480; WO 01/14417; and WO 00/66742. Plant-optimized genes thatencode such proteins, wherein the genes are engineered to use codons foroptimized expression in plants, have also been disclosed. See e.g. U.S.Pat. No. 6,218,188 and WO 00/24904.

BRIEF SUMMARY OF THE INVENTION

This invention provides truncated Cry35 proteins that surprisingly andunexpectedly have increased pesticidal activity as compared tofull-length Cry35 proteins. The subject invention also includespolynucleotides that encode these truncated proteins, transgenic plantscomprising a truncated gene of the subject invention, and transgenicplants that produce these truncated proteins. This invention furtherprovides methods of controlling plant pests, including rootworms, withthese truncated proteins. The truncated Cry35 proteins of the subjectinvention are preferably used in combination with Cry34 proteins, whichare known in the art. Various surprising advantages of the subjectinvention will be apparent in light of this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment of Cry35Aa1 (80JJ1), Cry35Ab1 (149B1),Cry35Ac1 (167H2), and 201L3 (˜45 kDa). The truncation site is in themiddle of the 4-leucine (L) run towards the C terminus. The amino acidsequence to be removed starts at and includes the third and fourth “Ls”to the C terminus.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of the wild-type 80JJ1 ˜45 kDa(Cry 35Aa1) protein.

SEQ ID NO:2 is the amino acid sequence of a preferred, truncated 80JJ1˜40 kDa (Cry 35Aa1) protein that exhibits enhanced toxin activity.

SEQ ID NO:3 is the amino acid sequence of the wild-type 149B1 ˜45 kDa(Cry 35Ab1) protein.

SEQ ID NO:4 is the amino acid sequence of a preferred, truncated 149B1˜40 kDa (Cry 35Ab1) protein.

SEQ ID NO:5 is the 4-leucine (L) run towards the C terminus, the middleof which is the truncation site.

SEQ ID NO:6 is the amino acid sequence of Cry35Ac1 (167H2) (GenBankAccession No. AAG50117) disclosed as SEQ ID NO:38 in WO 01/14417.

SEQ ID NO:7 is the amino acid sequence of 201L3 (˜45 kDa) disclosed asSEQ ID NO:136 in WO 01/14417.

DETAILED DESCRIPTION

This invention provides truncated Cry35 proteins that surprisingly andunexpectedly have increased pesticidal activity as compared tofull-length Cry35 proteins. Preferred proteins of the subject inventionare approximately 40 kDa. Wild-type Cry35 proteins are typically about45 kDa.

The subject invention also includes polynucleotides that encode thesetruncated proteins, transgenic plants comprising a truncated gene of thesubject invention, and transgenic plants that produce these truncatedproteins. Seeds and other materials (such as pollen and germ plasm)produced by such plants are also included within the subject invention.This invention further provides methods of controlling plant pests,including rootworms, by contacting the pests with these truncatedproteins. The truncated Cry35 proteins of the subject invention arepreferably used in combination with Cry34 proteins, which are known inthe art. Various surprising advantages of the subject invention will beapparent in light of this disclosure.

While some B.t. proteins are known to undergo protealytic processing invivo, there was no reason to expect the subject truncations to havebetter activity than the wild-type form. Thus, there was no priormotivation or suggestion to use the subject truncated genes in plants.Likewise there was no motivation or suggestion to provide (i.e.,directly administer) a truncated Cry35 protein to a pest so that thepest would ingest/consume the truncated protein.

Polynucleotides of the subject invention preferably have codon usagethat is optimized for expression in plants. Various techniques for doingso are well-known in the art.

The subject invention includes methods of controlling and/or inhibitingpests, preferably a rootworm pest, wherein the methods includescontacting the pest with a protein of the subject invention. Preferably,the truncated protein of the subject invention is produced by and ispresent in a transgenic plant comprising a gene that encodes thetruncated protein. By consuming material of this transgenic plant, suchas root cells of such plant, the pest thereby contacts the subjectproteins, which is preferably in the plant cells consumed by the pest.

Proteins of the subject invention having “toxin activity,” as the termis used herein, include proteins that enhance or improve the activity ofother toxin proteins. Cry35 proteins are known to act with Cry34proteins to exert the toxic effects. There are some reports that Cry34proteins can be toxic alone, but that toxicity is much improved whenused with Cry35 proteins. The subject invention provides a verysurprising means for still further increasing the Cry34/35 activity.

When an insect comes into contact with an effective amount of a “toxin”of the subject invention delivered via transgenic plant expression,formulated protein composition(s), sprayable protein composition(s), abait matrix or other delivery system, the results are typically death ofthe insect, inhibition of the growth and/or proliferation of the insect,and/or prevention of the insects from feeding upon the source(preferably a transgenic plant) that makes the toxins available to theinsects. Complete lethality to feeding insects is preferred, but is notrequired to achieve toxin activity. If an insect avoids the toxin orceases feeding, that avoidance will be useful in some applications, evenif the effects are sublethal or lethality is delayed or indirect. Forexample, if insect resistant transgenic plants are desired, thereluctance of insects to feed on the plants is as useful as lethaltoxicity to the insects because the ultimate objective is avoidinginsect-induced plant damage. Insects inhibited by the methods andproteins of the subject invention include those that are killed by thesubject methods and proteins.

Without being bound by any specific theory of a mechanism of action, andnoting that the following theory is now possible in light of the subjectdisclosure, the removal of the C terminus could facilitate assembly oftruncated Cry35 proteins into a multimer (comprised of truncated, Cry35monomers). There are other proteins having a motif where an activationdomain is protealytically removed to allow assembly of multimers. Notingthat the Cry35 protein is known to act with the Cry34 (˜14 kDa) protein,the Cry34 protein could bind to the multimeric form of assembled Cry35proteins. This could facilitate entry of the 14 kDa protein, which mayhave a cellular target via binding, or may form pores on its own. Itappears unlikely that a membrane-bound Cry35 monomer associates with themembrane and then with the 14 kDa as a binding partner.

While the 149B1 protein is specifically exemplified, any other cry35gene or protein can be used according to the subject invention, as Cry35proteins have similar structures and features. Thus, as one skilled inthe art would know, with the benefit of this disclosure, correspondingresidues and segments are now identifiable in the other Cry35 proteins.Thus, the specific examples disclosed herein can be applied to the otherproteins in the Cry35 family. The exact numbering of the residues mightnot strictly correspond to the 149B1 protein, but the correspondingresidues are readily identifiable in light of the subject disclosure. Inthis regard, some sequences of various Cry35 proteins and genesdescribed in various patent references are indicated below (suchsequences can be used according to some embodiments of the subjectinvention):

Cry designation Source isolate GENBANK Acc. No. 35Aa1 PS80JJ1 AAG5034235Aa2 EG5899 AAK64561 35Ab1 PS149B1 AAG41672 35Ab2 EG9444 AAK64563 35Ac1PS167H2 AAG50117 35Ba1 EG4851 AAK64566

35Aa1, 35Ab1, and 35Ac1 are also disclosed in WO 01/14417 as follows.The location (amino acid residue numbers) of the four leucine run (LLLL)(SEO ID NO:5) is also indicated.

SEQ ID NO: Location of LLLL Source isolate IN WO 01/14417 Length (SEQ IDNO:5) PS80JJ1 11 385 353-356 PS167H2 38 383 353-356 PS149B1 43 383353-356

There are many additional Cry35 sequences disclosed in WO 01/14417 thatcan be used according to the subject invention. For example:

SEQ ID NO: Location of LLLL Source isolate IN WO 01/14417 (SEQ ID NO:5)PS131W2 54 353-356 PS158T3 58 353-356 PS185FF 64 353-356 PS185GG 68353-356 PS187F3 78 353-356 PS187L14 86 353-356 PS187Y2 90 340-344 PS69Q116 353-356 KR589 126 353-356 PS201L3 136 355-358 PS187G1 140 353-356PS201HH2 144 (partial sequence) KR1369 148 353-356

Several other source isolates are also disclosed in WO 01/14417. The PSdesignation of the source isolate can be dropped for ease of referencewhen referring to a protein obtainable from that isolate. For example,it can be noted that the full-length 201L3 Cry35 protein is 387 residueslong. Polynucleotides that encode various Cry35 proteins are alsodisclosed in various references cited herein and are known in the art.

As shown by the above tables, the typical and preferred site fortruncation is after residue 354 for most Cry35 proteins. Thus, preferredtruncated Cry35 proteins consist of 354 amino acid residues.Accordingly, in preferred embodiments, approximately 29 residues areremoved from the C terminus of the wild-type/full-length Cry35 protein.However, approximately 31 residues (in the case of 80JJ1, for example)or 33 residues (in the case of 201L3, for example) can be removed fromthe C terminus. Some preferred proteins of the subject invention can be356 amino acid residues in length (in the case of 201L3, for example).

With that noted, truncated Cry35 proteins of the subject invention canbe part of a larger fusion protein. For example, an approximately354-residue, truncated Cry35 protein can be fused to a Cry34 protein tomake a Cry35/Cry34 chimeric protein. Thus, such proteins can be said tocomprise approximately 354 (or 356) Cry35 residues. A Cry35/Cry34 fusionis disclosed in WO 01/14417 as SEQ ID NO:159, with LLLL (SEQ ID NO:5)occurring at residue positions 353-356.

Fragments of full-length genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes that encode activated fragments of the subject invention canbe obtained using a variety of restriction enzymes. Proteases may beused to directly obtain activated fragments of the subject invention.

The polynucleotides of the subject invention can be used to formcomplete “genes” to encode proteins or peptides in a desired host cell.For example, as the skilled artisan would readily recognize, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art.

As used herein, reference to “isolated” and/or “purified” proteinsand/or polynucleotides indicates that such molecules are not in theirnative state. Thus, reference to “isolated” and/or “purified” signifiesthe involvement of the “hand of man” as described herein. For example, abacterial toxin “gene” of the subject invention put into a plant forexpression is an “isolated polynucleotide.” Likewise, a protein of thesubject invention that is produced by a plant is an “isolated protein.”

Because of the degeneracy/redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate alternative DNA sequences that encode the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention.

Certain toxins of the subject invention have been specificallyexemplified herein. As these toxins are merely exemplary of the proteinsof the subject invention, it should be readily apparent that the subjectinvention comprises variant or equivalent proteins (and nucleotidesequences coding for equivalents thereof) having the same (or similar),increased toxin activity of the exemplified proteins. Genes and toxinsof the subject invention include not only the specifically exemplifiedsequences, but also variants, mutants, chimerics, and fusions thereof.Proteins of the subject invention can have substituted amino acids solong as they retain the approximate size and the increased toxinactivity of the truncated proteins exemplified herein. As used herein,reference to a “variant” or an “equivalent” sequence refers to sequenceshaving amino acid substitutions, deletions, additions, as compared tothe wild-type sequence corresponding to a truncation of the subjectinvention, so long as the increased toxin activity exhibited by thetruncated protein is retained. Thus, truncated proteins of the subjectinvention include not only those having a wild-type sequence that istruncated. Some residues can be substituted. While conservative changesare preferred, nonconservative changes can also be made in some cases.Techniques for producing and confirming the activity of proteinsmodified accordingly are well-known in the art.

Equivalent toxins and/or genes encoding these equivalent toxins can beobtained/derived from wild-type or recombinant bacteria and/or fromother wild-type or recombinant organisms using the teachings providedherein. Other Bacillus and Paenibacillus species, for example, can beused as source isolates.

Variations of genes may be readily constructed using standard techniquesfor making point mutations, for example. In addition, U.S. Pat. No.5,605,793, for example, describes methods for generating additionalmolecular diversity by using DNA reassembly after random fragmentation.Variant genes can be used to produce variant proteins; recombinant hostscan be used to produce the variant proteins. Using these “geneshuffling” techniques, equivalent genes and proteins can be constructedthat comprise any 5, 10, or 20 contiguous residues (amino acid ornucleotide) of any sequence exemplified herein. As one skilled in theart knows, the gene shuffling techniques can be adjusted to obtainequivalents having, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, or 357 contiguous residues (amino acid or nucleotide) of anyof the exemplified or suggested sequences (or the complements (fullcomplements) thereof). Full-length genes obtained by shuffling can thenbe truncated according to the subject invention (or truncated genes canbe subjected to shuffling).

Variant/equivalent, truncated (and activated) toxins included within thescope of the subject invention will be of a length suggested herein andwill have some level of amino acid identity, similarity, and/or homologywith an exemplified activated toxin. This degree of amino acid identitywith an exemplified, truncated toxin is preferably greater than 60%,preferably greater than 75%, more preferably greater than 80%, even morepreferably greater than 90%, and can be greater than 95%. Preferredpolynucleotides and proteins of the subject invention can also bedefined in terms of more particular identity and/or similarity ranges.For example, this identity and/or similarity can be 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified or suggested herein.

Unless otherwise specified, as used herein, percent sequence identityand/or similarity of two sequences is determined using the algorithm ofKarlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402-410.BLAST nucleotide searches are performed with the NBLAST program,score=100, wordlength=12. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is used as described in Altschul et al. (1997),Nucl. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (NBLAST andXBLAST) are used. See NCBI/NIH website. The scores can also becalculated using the methods and algorithms of Crickmore et al. asdescribed in the Background section, above.

The amino acid homology/similarity/identity can be highest in criticalregions of the protein that account for its toxin activity or that areinvolved in the determination of three-dimensional configurations thatare ultimately responsible for the toxin activity. In this regard,certain amino acid substitutions are acceptable and can be tolerated.For example, these substitutions can be in regions of the protein thatare not critical to activity. Analyzing the crystal structure of aprotein, and software-based protein structure modeling, can be used toidentify regions of a protein that can be modified (using site-directedmutagenesis, shuffling, etc.) to actually change the properties and/orincrease the functionality of the protein.

In many cases, conservative amino acid substitutions can be made. Unlessotherwise specified, amino acids can be placed in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby an amino acid of one class is replaced withanother amino acid of the same type are within the scope of the subjectinvention so long as the substitution is not adverse to the elevatedtoxin activity exhibited by truncated proteins of the subject invention.Table 1 provides a listing of examples of amino acids belonging to eachclass.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the functional/biological/toxin activity of the protein.

In other embodiments, polynucleotides (and the proteins they encode) ofthe subject invention can be defined by their ability to hybridize withexemplified and/or suggested polynucleotides. The nucleotide sequencesof the subject invention are particularly advantageous, and they encodeproteins that are distinct from previously described proteins. Thus,exemplified or suggested polynucleotides (and/or the complementsthereof) can be used as hybridization probes.

As the skilled artisan knows, DNA typically exists in a double-strandedform. In this arrangement, one strand is complementary to the otherstrand and vice versa. As DNA is replicated in a plant (for example),additional complementary strands of DNA are produced. The “codingstrand” is often used in the art to refer to the strand that binds withthe anti-sense strand. The mRNA is transcribed from the “anti-sense”strand of DNA. The “sense” or “coding” strand has a series of codons (acodon is three nucleotides that can be read as a three-residue unit tospecify a particular amino acid) that can be read as an open readingframe (ORF) to form a protein or peptide of interest. In order toproduce a protein in vivo, a strand of DNA is typically transcribed intoa complementary strand of mRNA which is used as the template for theprotein. Thus, the subject invention includes the use of the exemplifiedpolynucleotides shown in the attached sequence listing and/orequivalents including the complementary strands. RNA and PNA (peptidenucleic acids) that are functionally equivalent to the exemplified DNAare included in the subject invention.

Polynucleotides of the subject invention, when accordingly used asprobes to define other polynucleotides (or proteins) that are within thescope of the subject invention, can be made detectable by virtue of anappropriate label or may be made inherently fluorescent as described inInternational Application No. WO 93/16094. The probes/polynucleotides ofthe subject invention may be DNA, RNA, or PNA. In addition to adenine(A), cytosine (C), guanine (G), thymine (T), and uracil (U; for RNAmolecules), synthetic probes (and polynucleotides) of the subjectinvention can also have inosine (a neutral base capable of pairing withall four bases; sometimes used in place of a mixture of all four basesin synthetic probes). Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “N” or “n” is usedgenerically, “N” or “n” can be G, A, T, C, or inosine. Ambiguity codesas used herein are in accordance with standard IUPAC naming conventionsas of the filing of the subject application (for example, R means A orG, Y means C or T, etc.).

As is well known in the art, if a probe molecule hybridizes with anucleic acid sample, it can be reasonably assumed that the probe andsample have substantial homology/similarity/identity. Preferably,hybridization of the polynucleotide is first conducted, followed bywashes under conditions of low, moderate, or high stringency bytechniques well-known in the art, as described in, for example, Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170. For example, as stated therein, low stringency conditionscan be achieved by first washing with 2×SSC (Standard SalineCitrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at roomtemperature. Two washes are typically performed. Higher stringency canthen be achieved by lowering the salt concentration and/or by raisingthe temperature. For example, the wash described above can be followedby two washings with 0.1×SSC/0.1% SDS for 15 minutes each at roomtemperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30minutes each at 55° C. These temperatures can be used with otherhybridization and wash protocols set forth herein and as would be knownto one skilled in the art (SSPE can be used as the salt instead of SSC,for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared bycombining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), andwater, adjusting pH to 7.0 with 10 N NaOH, then adjusting the volume to1 liter 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml ofautoclaved water, then diluting to 100 ml.

Detection of the probe provides a means for determining in a knownmanner whether hybridization has been maintained. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein. That is, one way to define a truncated gene of the subjectinvention is by the size of the protein it encodes (i.e., the protein orpolypeptide segment will be of a length disclosed or suggested herein)and by its ability to hybridize (under any of the conditionsspecifically disclosed herein) with an exemplified or suggestedsequence, such as a polynucleotide encoding a truncated protein of SEQID NO:2 and/or SEQ ID NO:4, for example. The same is true for apolynucleotide encoding a truncated 158×10 Cry35 protein, for example,of the subject invention.

As used herein, “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes was performed by standardmethods (see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1982]Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). In general, hybridization and subsequentwashes were carried out under conditions that allowed for detection oftarget sequences. For double-stranded DNA gene probes, hybridization wascarried out overnight at 20-25° C. below the melting temperature (Tm) ofthe DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/mldenatured DNA. The melting temperature is described by the followingformula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, andF. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K.Moldave [eds.] Academic Press, New York 100:266-285):Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula:Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura,and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes,D. D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

-   -   Low: 1 or 2×SSPE, room temperature    -   Low: 1 or 2×SSPE, 42° C.    -   Moderate: 0.2× or 1×SSPE, 65° C.    -   High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following is an example that illustrates procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE Improved Efficacy of Truncated Cry35Ab1

The native intact Cry35Ab1 protein from B.t. PS149B1 is approximately 44kDa. This 44-kDa protein (intact or full-length form), when expressed inthe bacteria and transgenic plants, is susceptible to cleavage byproteases producing a stable truncated form. The truncated Cry35Ab1 hasa molecular weight of 40.3-kDa determined by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).It was determined that both the intact and truncated forms possess thesame N-terminal sequence, indicating that the truncation occurred at theC-terminal domain. The results of the molecular mass determined byMALDI-TOF MS, together with data of N-terminal sequencing, suggest thatthe 40.3-kDa truncated Cry35Ab1 protein contains amino acids residues of1-354 of the intact Cry35Ab1, i.e., the cleavage site is between aminoacid residue #354 and #355 (L³⁵³L^(354↓)L³⁵⁵L³⁵⁶) (SEQ ID NO:5). Thiscleavage site was further confirmed by the data of C-terminal sequenceobtained with PSD (post source decay) mode of MALDI-TOF MS andcarboxypeptidase digestion techniques.

As displayed in FIG. 1, the sequence segment of the 4 L (leucine) isquite conserved among the Cry35 protein family. This cleavage siteappears to be universal to the whole family of Cry35 proteins.

The truncated version of the Cry35Ab1 protein was found to be moreefficacious than the non-truncated version in terms of synergizing theCry34Ab1 protein. This same efficacy improvement can be effected intransgenic plants and with homologous proteins.

Ten diet-overlay assays were conducted against neonate southern cornrootworm, Diabrotica undecimpuctata howardi. Purified full-length andtruncated Cry35Ab1 proteins were each applied at 0.5 μg/cm² incombination with Cry34Ab1 at 0.5 μg/cm². Controls consisted of Cry34Ab1alone applied at 0.5 μg/cm², and a buffer blank. Sixteen insects wereincluded for each treatment for each of ten bioassays. Insect weightswere collected for each group of 16 insects in each bioassay after sixdays of exposure to the treated diet. Statistical analysis consisted ofANOVA analysis of logarithmically transformed insect weights followed bymean comparisons using Duncan's Multiple Range Test. The logarithmictransformation was used to improve the homogeneity of variance. A secondANOVA of the raw weights was also carried out for the two Cry35Ab1treatments alone where good homogeneity of variance was seen. Meaninsect weights for the buffer control, Cry34Ab1 alone, full-lengthCry35Ab1+Cry34Ab1, and truncated Cry35Ab1+Cry34Ab1 treatments were 15.7,11.9, 8.8, and 6.9 mg/16 insects, respectively. Significant differencesin insect weight were seen between all treatments. The concentrations ofthe mixtures were based on a model generated using a preparation ofCry35Ab1 that contained a mixture of full-length and truncated protein(Herman, R. A., Scherer, P. N., Young, D. L., Mihaliak, C. A., Meade,T., Woodsworth, A. T., Stockhoff, B. A., and Narva, K. E., 2002, “BinaryInsecticidal Crystal Protein from Bacillus thuringiensis strain PS149B1:effects of individual protein components and mixtures in laboratorybioassays,” J. Econ. Entomol. 95:635-639). This model predicts 50%growth inhibition (GI) at the tested concentrations. The full-lengthprotein mixture in the tests reported here averaged 44% GI, while thetruncated protein mixture averaged 56% GI. This improvement in GItranslates to a ten-fold improvement in potency based on the modeldescribed in Herman et al. (2002). This analysis thus demonstrates animprovement in the activity of the truncated Cry35Ab1 protein comparedto the full-length form.

1. A polynucleotide that encodes an isolated, truncated Cry35 protein having a truncated C terminus and improved toxin enhancing activity against an insect, as compared to a full-length Cry35 molecule, wherein said protein enhances toxin activity of a Cry34 molecule, said protein has 354 amino acid residues, said protein has leucine-leucine residues as its C-terminal amino acids, and said protein has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
 2. The polynucleotide of claim 1, wherein said protein has a molecular weight of approximately 40 kDa and said Cry35 molecule has a molecular weight of approximately 45 kDa.
 3. The polynucleotide of claim 1, wherein said protein is fused to a Cry34 polypeptide.
 4. A polynucleotide that encodes a protein that consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
 5. The polynucleotide of claim 1, wherein any amino acid changes are conservative substitutions.
 6. The polynucleotide of claim 1, wherein said insect is a corn rootworm.
 7. The polynucleotide of claim 1, wherein said polynucleotide has a codon composition that is optimized for expression in plants.
 8. A plant comprising the polynucleotide of claim
 1. 9. A transgenic plant cell or bacterial cell comprising the polynucleotide of claim
 1. 10. A method of inhibiting a rootworm wherein said method comprises providing a Cry34 protein and a truncated Cry35 protein encoded by the polynucleotide of claim 1 to said rootworm for ingestion, wherein said Cry34 protein and said truncated Cry35 protein are produced by and are present in a plant.
 11. A plant expression vector comprising the polynucleotide of claim
 1. 